Enhancing Immunity to Tuberculosis

ABSTRACT

The invention is directed to compositions and methods for generating or enhancing the immune system of a patient against infection by a pathogen, and in particular MTB. Compositions of the invention contain one or more non-naturally occurring antigens that generate an effective cellular or humoral immune response to MTB and/or antibodies that are specifically reactive to mycolic acid or to the surface of MTB. The greater activity of the immune system generated by a vaccine of the invention involve an conjugation of peptides to increase in the generation of memory T cells that provide for a greater and/or longer lived or extended response to an MTB infection. Preferably a response involves an increased generation of antibodies that enhance immunity against MTB infection and promote an enhanced phagocytic response.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/872,391 entitled “Enhancing Immunity to Tuberculosis” filed Aug. 30,2013, the entirety of which is hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 28, 2014, isnamed 3022.023.PCT_SL.txt and is 3,682 bytes in size.

BACKGROUND

1. Field of the Invention

The present invention is directed to compositions and methods fortreating a disease or disorder and/or enhancing the immune system of apatient and, in particular, vaccines of non-naturally occurringsubstances and vaccination methods for treating and/or enhancing theimmune system against infection by Mycobacterium tuberculosis.

2. Description of the Background

Mycobacterium tuberculosis (MTB) is a pathogenic bacterial species inthe family Mycobacteriaceae and the causative agent of most cases oftuberculosis (TB). Another species of this genus is M. leprae, thecausative agent of leprosy. MTB was first discovered in 1882 by RobertKoch, M. tuberculosis has an unusual, complex, lipid rich, cell wallwhich makes the cells impervious to Gram staining. Acid-fast detectiontechniques are used to make the diagnosis instead. The physiology of M.tuberculosis is highly aerobic and requires significant levels of oxygento remain viable. Primarily a pathogen of the mammalian respiratorysystem, MTB is generally inhaled and, in five to ten percent ofindividuals, will progress to an acute pulmonary infection. Theremaining individuals will either clear the infection completely or theinfection may become latent. It is not clear how the immune systemcontrols MTB, but cell mediated immunity is believed to play a criticalrole (Svenson et al., Human Vaccines, 6-4:309-17, 2010). Commondiagnostic methods for TB are the tuberculin skin test, acid-fast stainand chest radiographs.

M. tuberculosis requires oxygen to proliferate and does not retaintypical bacteriological stains due to high lipid content of its cellwall. While mycobacteria do not fit the Gram-positive category from anempirical standpoint (i.e., they do not retain the crystal violetstain), they are classified as acid-fast Gram-positive bacteria due totheir lack of an outer cell membrane.

M. tuberculosis has over one hundred strain variations and divides every15-20 hours, which is extremely slow compared to other types of bacteriathat have division times measured in minutes (Escherichia coli candivide roughly every 20 minutes). The microorganism is a small bacillusthat can withstand weak disinfectants and survive in a dry state forweeks. The cell wall of MTB contains multiple components such aspeptidoglycan, mycolic acid and the glycolipid lipoarabinomannan. Therole of these moieties in pathogenesis and immunity remaincontroversial. (Svenson et al., Human Vaccines, 6-4:309-17, 2010).

When in the lungs, M. tuberculosis is taken up by alveolar macrophages,but these macrophages are unable to digest the bacteria because the cellwall of the bacteria prevents the fusion of the phagosome with alysosome. Specifically, M. tuberculosis blocks the bridging molecule,early endosomal autoantigen 1 (EEA1); however, this blockade does notprevent fusion of vesicles filled with nutrients. As a consequence,bacteria multiply unchecked within the macrophage. The bacteria alsocarry the UreC gene, which prevents acidification of the phagosome, andalso evade macrophage-killing by neutralizing reactive nitrogenintermediates.

The BCG vaccine (Bacille de Calmette et Guérin) against tuberculosis isprepared from a strain of the attenuated, but live bovine tuberculosisbacillus, Mycobacterium bovis. This strain lost its virulence to humansthrough in vitro subculturing in Middlebrook 7H9 media. As the bacteriaadjust to subculturing conditions, including the chosen media, theorganism adapts and in doing so, loses its natural growthcharacteristics for human blood. Consequently, the bacteria can nolonger induce disease when introduced into a human host. However, theattenuated and virulent bacteria retain sufficient similarity to provideimmunity against infection of human tuberculosis. The effectiveness ofthe BCG vaccine has been highly varied, with an efficacy of from zero toeighty percent in preventing tuberculosis for duration of fifteen years,although protection seems to vary greatly according to geography and thelab in which the vaccine strain was grown. This variation, which appearsto depend on geography, generates a great deal of controversy over useof the BCG vaccine yet has been observed in many different clinicaltrials. For example, trials conducted in the United Kingdom haveconsistently shown a protective effect of sixty to eighty percent, butthose conducted in other areas have shown no or almost no protectiveeffect. For whatever reason, these trials all show that efficacydecreases in those clinical trials conducted close to the equator. Inaddition, although widely used because of its protective effects againstdisseminated TB and TB meningitis in children, the BCG vaccine islargely ineffective against adult pulmonary TB, the single mostcontagious form of TB.

A 1994 systematic review found that the BCG reduces the risk of gettingTB by about fifty percent. There are differences in effectiveness,depending on region due to factors such as genetic differences in thepopulations, changes in environment, exposure to other bacterialinfections, and conditions in the lab where the vaccine is grown,including genetic differences between the strains being cultured and thechoice of growth medium.

The duration of protection of BCG is not clearly known or understood. Inthose studies showing a protective effect, the data are inconsistent.The MRC study showed protection waned to 59% after 15 years and to zeroafter 20 years; however, a study looking at Native Americans immunizedin the 1930s found evidence of protection even 60 years afterimmunization, with only a slight waning in efficacy. Rigorous analysisof the results demonstrates that BCG has poor protection against adultpulmonary disease, but does provide good protection against disseminateddisease and TB meningitis in children. Therefore there is a need for newvaccines and vaccine antigens that can provide solid and long-termimmunity to MTB.

The role of antibodies in the development of immunity to MTB iscontroversial. Current data suggests that T cells, specifically CD4⁺ andCD8⁺ T cells, are critical for maximizing macrophage activity againstMTB and promoting optimal control of infection (Slight et al, JCI123(2):712, February 2013). However, these same authors demonstratedthat B cell deficient mice are not more susceptible to MTB infectionthan B cell intact mice suggesting that humoral immunity is notcritical. Phagocytosis of MTB can occur via surface opsonins, such asC3, or nonopsonized MTB surface mannose moieties. Fc gamma receptors,important for IgG facilitated phagocytosis, do not seem to play animportant role in MTB immunity (Crevel et al., Clin Micro Rev. 15(2),April, 2002; Armstrong et al., J Exp Med. 1975 Jul. 1; 142(1):1-16). IgAhas been considered for prevention and treatment of TB, since it is amucosal antibody. A human IgA monoclonal antibody to the MTB heat shockprotein HSPX (HSPX) given intra-nasally provided protection in a mousemodel (Balu et al, J of Immun. 186:3113, 2011). Mice treated with IgAhad less prominent MTB pneumonic infiltrates than untreated mice. Whileantibody prevention and therapy may be hopeful, the effective MTBantigen targets and the effective antibody class and subclasses have notbeen established (Acosta et al, Intech, 2013).

Cell wall components of MTB have been delineated and analyzed for manyyears. Lipoarabinomannan (LAM) has been shown to be a virulence factorand a monoclonal antibody to LAM has enhanced protection to MTB in mice(Teitelbaum, et al., Proc. Natl. Acad. Sci. 95:15688-15693, 1998,Svenson et al., Human Vaccines, 6-4:309-17, 2010). The mechanism wherebythe MAB enhanced protection was not determined and the MAB did notdecrease bacillary burden. It was postulated that the MAB possiblyblocked the effects of LAM induced cytokines. The role of mycolic acidfor vaccines and immune therapy is unknown. It has been used fordiagnostic purposes, but has not been shown to have utility for vaccineor other immune therapy approaches. While MTB infected individuals maydevelop antibodies to mycolic acid, there is no evidence that antibodiesin general, or specifically mycolic acid antibodies, play a role inimmunity to MTB.

Antibiotic resistance is becoming more and more of a problem fortreating MTB infections. The BCG vaccine against TB does not provideprotection from acquiring TB to a significant degree. Thus there is astrong need to provide or improve products and approaches to prevent andtreat MTB.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provide new tools andmethods for enhancing the immune system.

One embodiment of the invention is directed to vaccines for thetreatment or prevention of infection of Mycobacterium tuberculosis (MTB)in a mammal comprising one or more non-naturally occurring antigens,which may be produced through recombinant techniques, preferablyincluding a pharmaceutically acceptable carrier. Preferably the antigencomprises an MTB surface secreted or intracellular antigen. Preferablythe antigen comprises one or more of a synthetic MTB peptide, syntheticMTB/influenza peptide composite, malaria, MTB surface antigen composite.A second approach utilizes non-natural moieties produced inalcohol-killed MTB, such as ethanol, heat-killed MTB orgluteraldehyde-killed MTB that generate an immune response against theone or more vaccine targets such as mycolic acid. The alcohol forexample denatures the proteins and disassociates the lipid structures inthe cell wall producing new and altered (non-natural) molecules.Preferably the pharmaceutically acceptable carrier comprises water, oil,fatty acid, carbohydrate, lipid, cellulose, or a combination thereof.Preferably peptides and antigen targets may be conjugated to proteinsand other moieties and delivered with adjuvants such as alum, squalineoil in water emulsion amino acids, proteins, carbohydrates and/or otheradjuvants.

Another embodiment of the invention is directed to methods for treatingor preventing MTB infection comprising administering the vaccine of theinvention to a mammal. Preferably the vaccine is administered to apatient orally, intramuscularly, intravenously or subcutaneously andgenerates a humoral response in the mammal that comprises generation ofantibodies specifically reactive against MTB moieties that impede hostimmunity or induce antibodies that enhance host immunity.

Another embodiment of the invention is directed to methods for treatingor preventing infection of Mycobacterium tuberculosis (MTB) in a mammalcomprising administering to the mammal polyclonal or monoclonalantibodies that are specifically reactive against MTB moieties, such asmycolic acid that stimulate cellular phagocytic activity and destructionof MTB by phagocytes, enhances cytokine induced immunity to MTB orneutralizes toxic MTB substances, and/or cocktails of two or moremonoclonal antibodies (MABs) that enhance immunity to MTB. Preferably,the anti-MTB antibodies are polyclonal antibodies or monoclonalantibodies and react against one or more MTB moieties.

Another embodiment of the invention is directed to monoclonal antibodiesthat are specifically reactive against mycolic acid of MTB. Preferablythe monoclonal antibody is an IgA, IgD, IgE, IgG or IgM, and may bederived from most any mammal such as, for example, rabbit, guinea pig,mouse, human, fully or partly humanized, chimeric or single chain of anyof the above. The DNA encoding the antibodies may be utilized in anyappropriate cell line to produce the encoded MABs. Another embodimentcomprises hybridoma cultures that produce the monoclonal antibodies.Another embodiment of the invention comprises non-naturally occurringpolyclonal antibodies that are specifically reactive against mycolicacid of MTB.

Another embodiment of the invention is directed to methods for treatingor preventing MTB infection by administering a monoclonal or polyclonalantibody that is specifically reactive against mycolic acid of MTB.

Another embodiment of the invention is directed to methods for treatingor preventing MTB infection by administering to a patient an effectiveamount of BCG vaccine and further enhancing the effectiveness and/or thelength of protection by also administering an effective amount of thevaccine of the invention that induces humoral immunity and providesenhanced phagocytic function. Enhanced phagocytic function by vaccine orantibody is defined as stimulated cellular phagocytic activity andenhanced destruction of the MTB bacillus inside the phagocyte.

Another embodiment of the invention is directed to methods ofidentifying one or more antibodies that activate phagocytizing cells,comprising: providing a microbe; generating antibodies that arespecifically responsive to the microbe: incubating the generatedantibodies with the phagocytizing cells; determining an activity of thephagocytizing cells after incubation with the antibodies; and selectingthe one or more antibodies that increase the activity of thephagocytizing cells as compared to a control. Preferably the microbe islive or killed MTB and optionally, the microbe can be treated with oneor more chemical and/or physical agents. Preferably the chemical agentis ethanol or gluteraldehyde. Also preferably, the antibodies generatedfrom a mouse and preferably monoclonal antibodies. Phagocytizing cellsinclude, but are not limited to macrophages, neutrophils, monocytes,mast cells, white blood cells, dendritic cells, phagocytic cell lines,HL-60 cells, U-937 cells, PMA treated cells, PMA treated U-937 cells,and combinations thereof. The activity of the cells can be determined,for example, by visual inspection, by antigen uptake, or fluorescentbased microscopy assay of the phagocytizing cells. Preferably thephagocytizing cells show activity only on incubation with the one ormore selected antibodies. Suitable controls include, for example, thephagocytic activity of the cells that have not been treated with anyantibodies, the phagocytic activity of the cells after incubation withantibodies provided against untreated antigen, or the phagocyticactivity of the cells after treatment with an agent that does notgenerate phagocytic activity. Preferably the one or more antibodiesselected treat or prevent microbe infection of a mammal. Alsopreferable, the one or more antibodies selected are mouse antibodiesthat have been humanized for the prevention and/or treatment of adisease or disorder.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 Antisera titers from M3 1319-1324 (Immunized with MTB non-naturalsurface antigens on the altered surface of EtOH-k TB) on EtOH-k TBcoating @ 1:1000.

FIG. 2 Antisera titers from M3 1325-1330 (Immunized with MTB non-naturalsurface antigens on the surface of Glut-k TB) on EtOH-k TB coating @1:1000.

FIG. 3 Antisera titers from M3 1331-1336 (Immunized with MTB non-naturalantigens from Son. Glut-k TB) on EtOH-k TB coating @ 1:1000.

FIG. 4 Antisera titers from M3 1337-1342 (Immunized with MTB non-naturalantigens from Son. Glut-k TB+adjuvant) on EtOH-k TB coating @ 1:1000.

FIG. 5 High level binding of isolated TB Pep01 (SEQ ID NO 1) at 1 μg/mland 10 μg/ml to MS 1124 sera at 42 days, 112 days and prefusion.

FIG. 6 Hybridoma productivity from MS 1143 and 1147 fusion as measuredon whole MTB (ethanol killed) and mycolic acid.

FIG. 7 Binding profiles of purified M1438 FEU II B3 (alpha-TB Pep 02) tovarious antigens (SEQ ID NO. 7).

DESCRIPTION OF THE INVENTION

Approximately one third of the world population is infected withMycobacterium tuberculosis (MTB). Current treatment includes a longcourse of antibiotics and often requires quarantining of the patient.Resistance is common and an ever increasing problem, as is the abilityto maintain the quarantine of infected patients. Present vaccinesinclude BCG which is prepared from a strain of attenuated(virulence-reduced) live bovine tuberculosis bacillus, Mycobacteriumbovis, and a live non-MTB organisms. BCG carries substantial associatedrisks, especially in immune compromised individuals, and has proved tobe only modestly effective and for limited periods. It is generallybelieved that a humoral response to infection by MTB is ineffective andoptimal control of infection must involve activation of T cells andmacrophages.

It has been surprisingly discovered that certain regions of MTB whenchemically or physically altered from their natural state generate anenhanced immune response against MTB in mammals. Preferred alterationsare created when the MTB is treated with chemicals such as, for example,ethanol, gluteraldehyde or another chemical that inactivates or killsthe organisms. In contrast, antigens of or antibodies generated againstthese regions without alteration (e.g. BCG vaccine) do not produce aprotective response even in adults with a robust immune system. Theseregions or epitopes that are created after treatment are referred to asimmunity enhancing antigens (IEAs). These IEAs are recognized by theimmune system of the host when administered to treat or preventinfection, by generating a cellular and/or humoral immune response tothe infection. Without limiting the invention, the non-naturallyoccurring IEAs of the invention are believed to be unrecognized by themammalian immune system due to physical changes created to the chemicalstructure of the antigen and/or by removal of one or more chemicalmoieties that otherwise block recognition of the epitope of the wholenon-altered MTB or even of a degradation product of the MTB organism. Onthe isolation of an IEA, the physical or chemical alteration of one ormore new epitopes are revealed to the host immune system generating aprotective response against infection that is not otherwise availablefrom a vaccine using whole or partial untreated organisms. Preferably,the IEAs of the invention are created from chemically killed organisms,such as ethanol killed, or degradation products of ethanol-killedorganisms. IEAs of MTB include, but are not limited to epitopic regionsof the surface of MTB, and various selected regions and sequences of theMTB components including, but not limited to MTB heat shock protein,peptidoglycan, mycolic acid and lipoarabinomannan (LAM). Preferred aminoacid and nucleic acid sequences of the invention contain or encode oneor more epitopes of an IEA for MTB, and/or additional epitopes specificfor other infections such as, for example, a viral infection (e.g.influenza). Preferred IEAs of the invention include altered portions ofpeptidoglycan, mycolic acid and LAM, which are useful as peptidevaccines and/or peptide adjuvants. Nucleic acid sequences of theinvention are preferably recombinantly produced and/or syntheticallymanufactured. Also preferred are nucleic acid aptamers and peptideaptamers and other molecules that mimic the structure and/or function ofthe non-natural antigens or antibodies of the invention. Also preferredare peptide and/or nucleic acid sequences that contain or encode one ormore epitopes of an IEA antigen of another pathogen, such as, forexample, a viral (DNA or RNA), bacterial, fungal or parasitic pathogenthat is the causative agent of a disease (e.g., influenza, HIV/AIDS,hepatitis, lower respiratory infections, measles, tetanus, cholera,malaria, viral and/or bacterial meningitis, infections of the digestivetract, pertussis, syphilis). Combinations of epitopes from both MTB andother pathogens include, for example, peptide conjugates of MTB andinfluenza or another viral epitope, peptide conjugates of MTB withDiphtheria toxin (e.g. CRM), Clostridium tetani toxin and peptides andproteins, or another bacterial epitope, or peptide conjugates of MTBwith Plasmodium falciparum or another parasitic epitope. Preferably, thepeptide sequences of the invention (e.g. see Table 3, which includespeptide composites of MTB, peptide composites of influenza, and combinedMTB-influenza composite peptides) are synthetic peptide vaccines thatgenerate and/or enhance an immune response to a pathogenic infectionsuch as, for example, MTB, influenza virus, or the etiological agents ofcholera, malaria, leprosy, AIDS, and/or another infectious disease, andprevent and/or treat the disease and infection. Also preferably, theimmune response generated is protective against the infection thatshields individuals from infection outside of the geographical or timeperiod of the limits of protection, for example, associated with thevarious BCG vaccines presently in use. Preferably, vaccines of theinvention provide protection to the patient for greater than about oneyear, more preferably greater than about two years, more preferablygreater than about three years, more preferably greater than about fiveyears, more preferably greater than about seven years, more preferablygreater than about ten years, and more preferably greater than aboutfifteen or twenty years.

Preferably the immune response generated upon the administration of avaccine of the invention is protective against TB or another infectionand enhance and/or prime the immune system of the patient to beimmunologically responsive to an infection such as by promotingrecognition of the pathogen, a greater and/or more rapid immunologicalresponse to an infection, phagocytosis of the pathogen or killing ofpathogen-infected cells, thereby promoting overall immune clearance ofthe infection. Preferably, a vaccination of an infected mammal with anIEA of the invention promotes the activation of a humoral and/orcellular response of the mammalian immune system For example,administering an IEA of the invention to an infected mammal promotes thesensing of the infection and then clears the infection from themammalian system by inducing or increasing phagocytic activity.Preferably this sensing and clearance activity is effective to clear thebody of both active organisms and latent or dormant organisms andthereby prevent a later resurgence of the infection.

One embodiment of the invention is directed to vaccines that, uponadministration to a patient, provide for protection against infection ofa pathogen. Vaccines containing IEAs are effective to stimulate acellular and/or humoral response in a patient. Alternatively the vaccinemay stimulate a humoral response that will stimulate an enhancedcellular or phagocytic cell response to any invading pathogen such asMTB. Preferably the vaccines of the invention contain an MTB EIA suchas, for example, one or more epitopes of altered peptidoglycan, mycolicacid, lipoarabinomannan (LAM), or a combination of one or more of thesealtered epitopes. Preferred MTB epitopes include MTB sequences andcomposites of MTB sequences plus other epitope sequence, such as thoselisted in Table 3.

Vaccines of the invention may contain one or multiple sequences and/orportions that are derived from the same or from different sourcematerials or organisms. Source materials include, for example, proteins,peptides, toxins, cell wall components, membrane components, polymers,carbohydrates, nucleic acids including DNA and RNA, lipids, fatty acids,and combinations thereof. Vaccines with multiple portions derived fromdifferent sources are referred to herein as conjugate vaccines and mayinclude portions derived from, for example, proteins and lipids,peptides and fatty acids, and lipids and nucleic acids. Vaccineconjugates may contain portions derived from distinct organisms, suchas, for example, any combination of bacteria (e.g. MTB), virus(preferably influenza, HIV, RSV), fungal or mold, and parasite (e.g.malaria). These conjugates may be composed of, for example, a portion ofmycolic acid of MTB coupled to serum albumin (e.g. bovine serum albuminor BSA). Exemplary conjugate vaccines include, but are not limited toconjugates of a surface protein of MTB, peptidoglycan, mycolic acid, orLAM with a protein such as tetanus toxin or diphtheria toxin.

Also preferred are vaccines of the invention that include one or more ofa pharmaceutically acceptable carrier, diluent, excipient, adjuvantand/or other medicinal or pharmaceutical agent or preparation known tothose skilled in the art. Preferred pharmaceutically carriers includeone or more of water, fatty acids, lipids, polymers, carbohydrates,gelatin, solvents, saccharides, buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents or an immunological inert substance, and especially preferred arecarriers designated as generally recognized as safe (GRAS) by the U.S.Food and Drug Administration or another appropriate authority.

Although the peptides of the invention may be complete vaccines againstan infection in and of themselves, it has also been discovered that thepeptide vaccines of the invention enhance the immune response whenadministered in conjunction with other vaccines against the same or asimilar infection such as, for example, BCG against a TB infection. As asecondary vaccine or adjunctive treatment in conjunction with anexisting primary vaccine treatment, secondary vaccines (which may beantibodies or antigens) of the invention provide a two punch defenseagainst infection which is surprisingly effective to prevent or extendthe period of protection available from the conventional primaryvaccine. The primary vaccine (i.e., conventional vaccine) and secondaryvaccines (vaccines of the invention) may be administered aboutsimultaneously, or in staggered fashion in an order determinedempirically or by one skilled in the art. Preferably the peptide vaccineis administered in advance of an attenuated or killed whole cellvaccine, but may also be administered after or simultaneously (e.g.,collectively as a single vaccination or as separate vaccinationcompositions). Preferably the peptide vaccine is administered frombetween about two to about thirty days in advance or afteradministration of the whole cell vaccine, and more preferably frombetween about four to about fourteen days in advance or after. Withoutbeing limited as to theory, it is currently believed that the firstvaccine primes the immune system of the subject and the second vaccineprovides the boost to the immune system creating a protectiveimmunological response in the patient.

Another embodiment of the invention comprises one or more antibodiesthat binds to one or more specific targets or pathogens, preferably oneor more MTB epitopes that are IEAs of the invention optionally includingone or more previously known epitopes. These antibodies, which may beeither monoclonal or polyclonal, have surprisingly demonstrated antigenbinding in ELISA assays to non-natural target MTB antigens, such asethanol altered MTB, and demonstrate enhanced immune response to MTB andpromote or enhance phagocytic clearance of MTB. Antibodies of thecurrent invention that stimulate phagocytic function promote phagocyteactivity to identify MTB, engulf the organism and then destroy the MTBbacilli. Antibodies enhance treatment, for example, by promotingphagocytosis of bacteria, stimulating T cell recognition of the foreignantigen (e.g. memory T cells) followed by cell-killing of infectedcells, and overall immune system clearance of the infection. Antibodiesof the invention have been developed to a number of antigen targets,including but not limited to mycolic acid of the surface of MTB,heat-shock proteins and other MTB antigens (e.g., 16 KD MTB heat-shockprotein of SEQ ID NO 1).

Another embodiment of the invention is directed to multiple antibodiesof the invention (polyclonal, monoclonal or fractions such as Fabfragments, single chains, etc.) that are combined or combined withconventional antibodies (polyclonal, monoclonal or fractions such as Fabfragments, single chains, etc.) into an antibody cocktail for thetreatment and/or prevention of an infection. Combinations can includetwo, three, four, five or many more different antibody combination witheach directed to a different antigen including IEAs of the invention.

Antibodies to one or more different IEAs of the invention may bemonoclonal or polyclonal and may be derived from any mammal such as, forexample, mouse, rabbit, pig, guinea pig, rat and preferably human.Polyclonal antibodies may be collected from the serum of infected orcarrier mammals (e.g., typically human, although equine, bovine,porcine, ovine or caprine may also be utilized) and preserved forsubsequent administration to patients with existing infections.Administration of antibodies for treatment against infection, whetherpolyclonal or monoclonal, may be through a variety of availablemechanisms including, but not limited to inhalation, ingestion, and/orsubcutaneous (SQ), intravenous (IV), intraperitoneal (ID), and/orintramuscular (IM) injection, and may be administered at regular orirregular intervals, or as a bolus dose.

Monoclonal antibodies to one or more IEAs of the invention may be of oneor more of the classes IgA, IgD, IgE, IgG, or IgM, containing alpha,delta, epsilon, gamma or mu heavy chains and kappa or lambda lightchains, or any combination heavy and light chains including effectivefractions thereof, such as, for example, single-chain antibodies,isolated variable regions, isolated Fab or Fc fragments, isolatedcomplement determining regions (CDRs), and isolated antibody monomers.Monoclonal antibodies to IEAs may be created or derived from human ornon-human cells and, if non-human cells, they may be chimeric MABs orhumanized. Non-human antibodies are preferably humanized by modifyingthe amino acid sequence of the heavy and/or light chains of peptides tobe similar to human variants, or genetic manipulation or recombinationof the non-coding structures from non-human to human origins. Theinvention further comprises recombinant plasmids and nucleic acidconstructions used in creating a recombinant vector and a recombinantexpression vector the expresses a peptide vaccine of the invention. Theinvention further comprises hybridoma cell lines created from the fusionof antibody producing cells with a human or other cell lines for thegeneration of monoclonal antibodies of the invention.

Antibodies to IEAs and other substances when recognized by the immunesystem, promote phagocytosis and clearing of an infection of thatmicroorganism and/or the development of immunity to infection.Pretreatment or simultaneous treatment of MTB with certain antibioticsexposes immune enhancing antigens of the microorganism to cell killingmechanisms of the immune system including, but not limited tophagocytosis, apoptosis, macrophage and natural-killer cell activation,cytokine and T-cell mediated cell killing, and complement-initiated celllysis.

Another embodiment of the invention is directed to methods foradministering to a patient a composition containing antibodies of theinvention and, preferably, with a pharmaceutically acceptable carrier.Antibodies to IEAs of a microorganism, either or both as polyclonalantibodies or monoclonal antibodies or cocktails of one or moreantibodies, may be administered individually and/or in combinations witheach other and/or other vaccines and/or treatments or preventions of themicroorganism infection. Antibodies to immune enhancing antigens may beadministered prophylactically prior to possible infection, or to treatan active or suspected MTB infection.

Preferably the vaccine of immune enhancing antigens and/or antibodies toimmune enhancing antigens of the invention is administered inconjunction with conventional vaccines against MTB (e.g., BCG) or as aPrime Boost with another vaccine such as, for example BCG. This combinedvaccine of the invention provides an enhancement of the immune responsegenerated and/or extends the effectiveness and/or length of period ofimmunity. Enhancement is preferably an increase in the immune responseto MTB infection such as an increase in the cellular or humoral responsegenerated by the host's immune system. An effective amount of vaccine,adjuvant and enhancing antigen of the invention is that amount whichgenerates an infection clearing immune response or stimulates phagocyticactivity. Upon administration of the combined vaccine, an increase ofthe cellular response may include the generation of targeted phagocytes,targeted and primed natural killer cells, and/or memory T cells that arecapable of maintaining and/or promoting an effective response toinfection for longer periods of time than the conventional vaccine wouldprovide alone. An increase in the humoral response may include thegeneration of a more diverse variety of antibodies including, but notlimited to different IgG isotypes or antibodies to more than one microbeor more than one MTB molecule that are capable of providing an effectiveresponse to prevent infection by MTB and/or another microbe as comparedto the humoral response that would be generated from just a conventionalMTB vaccine. Administration preferably comprises combining BCG vaccineand a vaccine antigen that generates a humoral response in the patientto a surface antigen of MTB. Preferably the response is to mycolic acid,peptidoglycan, lipoarabinomannan and/or another component of themicroorganism, preferably one that presents or is otherwise exposed onthe surface of MTB or secreted during infection. Some substancesproduced by MTB may be toxic to the host immune system or impede immunefunction. Antibodies that clear or neutralize these toxic substances(such as released or free mycolic acid components) can further act toenhance and improve host immunity.

Exposure of these MTB antigens to the antibodies of the invention or ofthe immune system of the patient may be augmented or substantiallyincreased by prior or about simultaneous treatment with individual orcombinations of antibiotics, cytokines and other bactericidal and/orbacteriostatic substances (e.g., substances that inhibit protein ornucleic acid synthesis, substances that injury membrane or othermicroorganism structures, substances that inhibit synthesis of essentialmetabolites of the microorganism), and preferably one or more antibioticor substance that attacks the cell wall structure or synthesis of thecell wall of the microorganism. Preferably, the antibiotics do not causethe release of cell surface antigens, but expose antigens that are nototherwise exposed or easily accessible to the immune system. Effectiveamounts of antibiotics are expected to be less than the manufacturerecommended amount or higher dose, but for short periods of time (e.g.about one hour, about 4 hours, about 6 hours, less than one or two day).Examples of such antibiotics include, but are not limited to one or moreof the chemical forms, derivatives and analogs of penicillin,amoxicillin, augmentin, polymyxin B, cycloserine, autolysin, bacitracin,cephalosporin, vancomycin, and beta lactam. Antibiotics worksynergistically with the immune enhancing antigens of the invention toprovide an efficient and effective preventative or treatment of aninfection. The antibiotics are not needed in bacteriostatic orbactericidal quantities, which is not only advantageous with regard toexpense, availability and disposal, these lower dosages do notnecessarily encourage development of resistance to the same degree,together a tremendous benefit of the invention. Preferably, theantibiotic is administered initially to damage and alter the pathogencell wall and epitopes (for example to produce a non-natural surface andexpose cell wall components such as mycolic acid non-natural epitopesand other moieties that can be recognized by the patient's immunesystem), followed a short time later with the antibody treatment, sothat the IEA is more fully accessible to the antibody when administered.The period of time between treatment may be one hour or more, preferably4 hours or more, preferably 8 hours or more, or preferably 12 or 24hours or more.

Antibodies to immune enhancing antigens of the invention may beadministered directly to a patient to treat or prevent infection of MTBvia inhalation, oral, SQ, IM, IP, IV or another effective route, oftendetermined by the physical location of the infection and/or the infectedcells. Treatment is preferably one in which the patient does not developor develops only reduced symptoms (e.g., reduced in severity, strength,period of time, and/or number) associated with MTB infection and/or doesnot become otherwise contagious. Antibodies used in conjunction withanti-MTB antibiotics will increase the clearance of MTB or inactivatesubstances that impede immunity as measured by a more rapid reduction ofsymptoms, more rapid time to smear negativity and improved weight gainand general health. In addition, treatment provides an effectivereduction in the severity of symptoms, the generation of immunity toMTB, and/or the reduction of infective period of time. Preferably thepatient is administered an effective amount of antibodies to prevent orovercome MTB infection alone or as adjunctive therapy with antibiotics.

Another embodiment of the invention is directed to methods ofidentifying one or more antibodies that activate phagocytizing cells.These methods comprise screening a population of antibodies and selectedthe one or more antibodies of those screened that are the effective inthe activation of phagocytizing cells. As a first step, microbes ofinterest are provided and may be purified, isolated or both. Themicrobes may be killed, attenuated or live microorganisms. Preferredmicrobes include MTB or another microorganism that cause an infectiousdisease in humans. Optionally, the microbe may be treated with achemical or physical agent and preferred treatment include, for example,exposure to a chemical such as ethanol or gluteraldehyde that alters thechemical structure of one or more antigens of the microbe, or physicalthat alters the microbe structure. Alteration can involve a chemicalchange, such as, for example, removal or alteration of a specificchemical moiety, or physical such for example a shifting of a moiety sothat a new region or epitope appears. The antibodies to be screened inthe methods of the invention can be created or generated, orcommercially provided. Preferably the antibodies are polyclonalantibodies, antibody fragments such as, for example, Fab, Fc and singlechain antibodies, or monoclonal derived from mice or another mammal. Theantibodies are next incubated with phagocytizing cells under conditionswhereby the activity of the cells can be measured during or after a setperiod of incubation. Activity can be any cellular activity such as, forexample, proliferation, the presence or absence of a marker, oxygenuptake or utilization, or determining any cellular activity such as,preferably, phagocytizing activity. Phagocytizing cells include most anycells that demonstrate phagocytosis and include for example,macrophages, neutrophils, monocytes, mast cells, white blood cells,dendritic cells, phagocytic cell lines, HL-60 cells, U-937 cells, PMAtreated cells, PMA treated U-937 cells, and combinations thereof. Themeasurement of activity can be performed by any technique known to thoseskilled in the art and is preferable by observation of the cells, by theappearance of cell vacuoles, by microbe or antigen uptake assays, or bymeasurement of phagocytizing markers. Preferably the measurement ofactivity is performed using a fluorescent-based microscopy assay. Upondetermining activity of phagocytizing cells incubated with theantibodies, one or more of the antibodies that showed activity or anincreased activity as compared to a control are selected. Controlsinclude, for example, phagocytic activity of the cells that have notbeen treated with any antibodies, the phagocytic activity of the cellsafter incubation with antibodies provided against untreated antigen, orthe phagocytic activity of the cells after treatment with an agent thatdoes not generate phagocytic activity. Preferably the activity ispresent only on incubation with antibodies specifically responsive tothe microbe. Selected antibodies are preferable useful for the treatmentand/or prevent of infection of the microbe. Preferably, when the microbeis MTB, the one or more antibodies that show increased activity ofphagocytizing cells as compared to a control can be used to treat and/orprevent MTB infection in a human or other mammal.

Although the invention is generally described in reference to humaninfection by Mycobacterium tuberculosis, as is clear to those skilled inthe art the compositions including many of the antibodies, tools andmethodology is generally and specifically applicable to the treatmentand prevention of many other diseases and infections in many othersubjects (e.g., cats, dogs, pets, etc.) and most especially diseaseswherein the causative agent is of viral, bacterial, fungal and parasiticorigins.

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

EXAMPLES Example 1

Mice bleeds: Female Balb/c mice were acquired at 3-4 weeks of age; 7-14days prior to the commencement of the study to allow them acclimate tothe facility. Thereafter, the mice were tagged with ear tags foridentification, and bled at the orbital lobe prior to immunization tohave a reference point. The mice were bled at days 20, 29, 63, and priorto fusion. About 150 μL-200 μL of blood was collected at each bleed.Antisera Titers for MS 1319-1342 Immunized with Washed Battelle Bugs(Batch III @ OD 600 nM=1.000).

Sera processing: At each bleed, blood was collected in micro-centrifugetubes and stored in cryo-vials at from 2-8° C. overnight. On the nextday, samples were centrifuged at 2000 rpm for 10 minutes at 22° C. Thetop layer of sera was carefully collected, avoiding red blood cells(RBC), and stored in new micro-centrifuge tubes at minus 20° C. In theevent that the sera could not be processed the next day, sera sampleswere processed on the same day as the bleed. Sera samples were placed ina 37° C. incubator for 30 minutes, and then placed at 2-8° C. for 15minutes. Afterwards, sera samples were centrifuged and processed in themanner indicated above. Sera processing was performed on the bench-top.

Killed MTB organisms: M. tuberculosis were grown in Middlebrook broth,washed three times in PBS and then suspended in either 70% ethanol or 2%glutaraldehyde activated with sodium bicarbonate. A third antigenpreparation was sonicated (Son), glutaraldehyde killed MTB. Washedethanol-killed and glutaraldehyde-killed MTB were obtained from Battelleat a concentration of 5.0×10⁸ CFU/mL. TB Pep 01 was produced by PiProteomics at a purity of over 90%.

Mice Immunizations:

Whole Bug Immunizations: Tuberculosis bacterial, strain Battelle (BatchIII), killed with ethanol (EtOH-k) or glutaraldehyde (Glut-k), werewashed in PBS to remove potential toxic substances. One mL of antigen atoriginal concentration was centrifuged at 12,000 rpm for 10 minutes. 900μL of the supernatant was discarded and the pellet re-suspended 900 μLof PBS by centrifugation at 12000 rpm for 10 minutes. This was repeatedtwo more times for a total of three washes. PBS was used because it isisotonic to blood and does not cause hardship to the mice.

Adjuvant Immunizations: 50% Alum and Titer-Max Gold (adjuvant). For thegroups with adjuvant Titer-Max Gold, the adjuvant comprised 60% of theinjection. Antigen was added to the adjuvant in a double plunger glasssyringe where the emulsion was prepared. The mice were immunized at day0 and boosted on Day-22, and within the week prior to fusion. Each mousewas immunized with 200 μL of antigen at varying concentrations to assessimmunogenicity. The immunizations were delivered subcutaneously, andthen intravenously prior to fusion. Enzyme-Linked Immunosorbent Assay(ELISA): The sera and supernatants (from hybridoma cells) were tested byELISA to determine antisera and hybridoma titers.

Fusion and Hybridoma Production: Post-Day 63, mice that had beenidentified by ELISA for high antisera titers were sacrificed and theirspleens harvested. The spleen cells were fused to SP2/0 myeloma cellsusing ethylene glycol, and 100 μl seeded and grown in sterile, 96-wellculture plates as adhesion cells. The fused cells were stored in a 37°C. humidified 5% CO₂ incubator. The fusion was performed in a sterilelaminar flow hood.

Cell Culture: On Day 1, the day after fusion, 1×HAT selection media wasintroduced to select for hybridoma cells. The cells were incubated at37° C. in a humidified 5% CO₂ incubator. On Day 9 or 10, they hybridomasupernatants were tested for antibody production. Afterwards, cells werefed twice a week, on Mondays and Fridays with hybridoma media thatconsisted of 15% FBS, 1% L-Glutamine, 0.1% Gentamycin, 1% Protein-freehybridoma media, and 1×HT media in DMEM. For each re-feed; 60 μl ofsupernatant were discarded and 100 μl of media added to each well. Thisprocess was performed using aseptic techniques in a sterile hood. Referto SOP-1005-00 Basic Cell Culture Techniques.

Mycolic Acid-BSA Conjugation:

Reagents: Mycolic acid from mycobacterium tuberculosis, Sigma Cat:M4537. N-hexane, Sigma Cat: 296090.1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride, TCI Cat:D1601. DMSO, Sigma Cat: D2650. Bovine serum albumin, Sigma Cat: A9418.

Method: 1.2 mg of mycolic acid was dissolved into 25 μL of n-hexane. 1.7mg of BSA was dissolved into 1.2 mL of 0.1M MES buffer pH 6, and 0.06 mLof DMSO was added with vortexing. The mycolic acid solution was addedslowly to the BSA solution with vortexing. 14 mg of EDC was added as drypowder with stirring. The pH was recorded to be 5.5 after all additionsand the reaction proceeded overnight at 4° C. The following day theconjugate solution was dialyzed against PBS-T in 14k MWCO tubing.

TB Peptide—Conjugation:

CRM-Flu Peptide 5906 (NS0243), CRM-TB peptide 1 (NS0245), CRM-TB peptide2 (NS0246) (see Table 1): CRM was brought to 6 mg/mL in 0.1M HEPES pH8+0.1% Tween 80. A 30 fold excess of 0.2M SBAP in DMSO was added whilevortexing and incubated at room temperature for 1 hour. Followingincubation, the CRM was dialyzed against PBS-EDTA pH 7.7. All peptideswere dissolved in 0.1M HEPES pH 8 at 10 mg/mL. A two fold molar excessof 0.2M SATA in DMSO was added while vortexing and the solutionsincubated at room temperature for one hour. The solutions were broughtto pH 6 with 1M sodium acetate and 1M NH₂OH was added to a finalconcentration of 50 mM. The CRM-SBAP was taken out of dialysis anddivided into 3×3 mg aliquots. The peptides were added to the CRM-SBAPwhile vortexing and the pH brought to 8 with 1M HEPES pH 8. Theconjugates were allowed incubate overnight at 4° C. The conjugates weredialyzed against PBS pH 8, put through a 0.2 μm filter, and the A₂₈₀ wasread for concentration using 1.07 as the 0.1% extinction coefficient ofCRM. CRM-Mycolic acid (NS0244): CRM was brought to 6 mg/mL in 0.1M HEPESpH 8+0.1% Tween 80. 5 mg of mycolic acid dissolved in 100 μL ofn-hexane. The CRM (3 mg) and 2 mg of mycolic acid were mixed and 50 mgof EDC was added. The solution had a final pH of 7.9 and incubatedovernight at 4° C. The conjugate dialyzed into PBS pH 8, filtered to 0.2μm, and the concentration was determined by A280.

TABLE 1 NS0243 NS0244 NS0245 NS0246 CRM 3 mg 3 mg 3 mg 3 mg Used Peptide3.6 mg 2 mg 4.5 mg 3.2 mg Used Final 2.3 0.64 2.4 1.84 OD Final 2.15mg/mL 0.6 mg/mL 2.24 mg/mL 1.72 mg/mL Concen- tration

Reagents: Tetanus toxoid obtained from the Serum Institute, Batch031L1006. Diphtheria toxoid (CRM) was obtained from Fina Biosolutions,Rockville, Md. DMSO, Sigma Cat: D2650. N-Succinimidyl3-(2-pyridyldithiol)-propionate (SPDP), Molecular BioSciences Cat:67432. 4-Maleimidobutyric aced NHS-ester (GMBS), Molecular BioSciencesCat: 98799. TB Peptide, PiProteomics, Name Peptide 1 (SEQ ID NO 1; the16 KD heat-shock MTB antigen “Promiscuous Peptide”) (Gowthaman et al.,JID 204: 1328-1338, 1 Nov. 2011). Dithiothreitol, Spectrum Cat: DI184.0.8 mg of peptide was diluted into 80 μL of 0.1M HEPES pH 8 and 19 μL of0.1M SPDP in DMSO was added with vortexing. In a separate vial, 5 mg ofBSA was diluted into 0.48 mL of 0.1M HEPES pH 7.4 and 7 μL of 0.1M GMBSin DMSO was added with vortexing. Both solutions were incubated at roomtemperature for 1 hour. The BSA-GMBS was dialyzed against 2 L ofPBS-EDTA pH 6.8. 1M DTT in NaOAc was added to the peptide solution to afinal concentration of 15 mM and incubated for 1 hour. The peptide wasdesalted on a P2 column with PBS-EDTA pH 6.8 and 0.2 mL fractions werecollected. The fractions were checked for 280 nm absorbance and thefirst half of the curve with 280 OD were pooled and added to theBSA-GMBS. The solution was allowed to react overnight at 4° C., followedby dialysis into PBS.

Example 2 Induction of Humoral Immunity

Mice immunized with MTB killed with ethanol (FIG. 1) or glutaraldehyde(FIG. 2) developed a strong humoral antibody response with good bindingto MTB. In addition, mice immunized with ethanol-killed MTB had a higherand more rapid rise in antibody titers than did mice immunized withGlut-killed MTB and SQ was superior to the IV route of immunization.Mice immunized SQ with sonicated MTB (FIG. 3) had increased antibodyresponses compared to IV and adjuvant, Alum and Tmax (squalene, wateroil emulsion) (FIG. 4), enhanced antibody to MTB in some mice. A summaryof the results from these experiments is provided in Table 2.

TABLE 2 ELISA Results Sample Route Mouse ID Prelim Day 21 Day 42 Day 63EtOH + TB SQ 1319 0.076 0.276 4.000 4.000 SQ 1320 0.074 0.763 3.8124.000 SQ 1321 0.076 0.519 4.000 4.000 IV 1322 0.063 1.553 3.346 3.611 IV1323 0.066 1.857 4.000 4.000 IV 1324 0.072 0.164 0.834 1.578 Glu + TB SQ1325 0.072 0.074 0.840 3.051 SQ 1326 0.062 0.060 0.272 0.588 SQ 13270.076 0.102 1.751 2.573 IV 1328 0.064 0.071 0.907 1.481 IV 1329 0.0940.081 0.106 0.135 IV 1330 0.086 0.240 0.561 0.915 Son/Glu + TB SQ 13310.085 0.193 1.722 2.752 SQ 1332 0.077 0.094 0.190 0.155 SQ 1333 0.0900.210 0.854 1.037 IV 1334 0.068 0.077 0.152 0.127 IV 1335 0.080 0.0770.097 0.096 IV 1336 0.062 0.070 0.085 0.135 Son/Glu + SQ 1337 0.0640.112 0.628 2.128 TB + Adjuvant SQ 1338 0.078 0.067 0.169 0.280 SQ 13390.071 0.096 0.356 2.422 IV 1340 0.092 0.101 0.185 0.149 IV 1341 0.0870.086 0.299 2.843 IV 1342 0.066 0.308 0.156 0.134

Mice immunized with ethanol killed TB had the best response and therewas little difference observed between immunizations SQ or IV. At day 21there was a significant difference in titers of SQ and IV immunizations.By day 42 and day 63, there was little to no difference.Glutaraldehyde-killed TB mice developed titers, but not until day 42 asthere appeared to be a delay to the immune response. Sonication wasthought to increase the availability of epitopes, but only 1331 and 1333(both SQ) developed titers at day 42 with an increase at day 63.Although adjuvant is supposed to increase activity of the immune system,the group with adjuvant had only modestly elevated titers at day 63. Onepossibility is that the epitopes did not respond effectively with thistype of adjuvant.

A strong binding to mycolic acid was demonstrated in post immunizationsera and further studies showed that when spleen cells were fused, themajority of MABs bound to MTB and mycolic acid. Mycolic acid impedesopsonophagoctosis and vaccines that induce humoral immunity to this cellwall component or antibodies that bind to this lipid would be useful toprevent or treat TB. A mycolic acid subunit vaccine or conjugate vaccinethat induces humoral immunity to MTB would be useful to prevent ormitigate TB infections.

Peptide Conjugate Vaccine

Mice immunized with a small TB peptide conjugate vaccine (SEQ ID NO 1)developed humoral immunity to this 16 KD heat shock protein. Theseantibodies to an important TB moiety provide another method for humoralimmune induction to mitigate against TB infection, either alone or withother antibodies raised against one or more other key targets, such asmycolic acid.

Example 3 Immunizations

Mouse 1124 was immunized with TB heat shock peptide-BSA conjugatevaccine (100 μg) on days 0, 21, 42 and 112. On day 152 (3 days beforesacrifice for splenic fusion), 6 logs of MTB that were ethanol killedwere injected IV. Priming with MTB peptides followed by whole MTBchallenge elicits a rapid rise to the priming peptide that can bedetected within 3 days (FIG. 5).

It is surprising that the titers were higher within 3 days of challengewith whole killed MTB. Also, although small, priming with MTB peptidesfollowed by whole MTB challenge elicited a rapid rise to the primingpeptide that could be detected within 3 days.

Example 4

Mice immunized with unwashed, ethanol killed MTB (as above), producednumerous hybridomas producing antibodies that bound to whole ethanolkilled MTB (FIG. 6). Surprisingly there was a close correlation betweenserum binding to Mycolic acid and MTB bacilli. This killed MTBimmunization produced a humoral immune response to mycolic acid and MTB,thus demonstrating that the polyclonal and monoclonal antibodies tomycolic acid, prepared according to the invention, can be useful forprevention and also treatment of MTB infections.

Example 5 Peptide Sequences

All peptides were synthetically manufactured. A listing of the sequencesand the epitopes contained within each peptide is shown in Table 3(Flu=influenza virus).

TABLE 3  Sequences of Peptides of VaccinesSEQ ID NO 1 SEFAYGSFVRTVSLPVGADE-TB Pep01SEQ ID NO 2 GNLFIAP (Flu HA epitope)SEQ ID NO 3 HYEECSCY (Flu NA epitope)SEQ ID NO 4 WGVIHHP (Flu HA epitope) SEQ ID NO 5 GNLFIAPWGVIHHPHYEECSCY(composite of Flu HA plus NA sequences)SEQ ID NO 6 WGVIHHPGNLFIAPHYEECSCY(composite of Flu HA plus NA sequences)SEQ ID NO 7 SEFAYGSFVRTVSLPVGADEGNLFIAPWGVIHHPHYEECSCY-TB Pep02 (composite of HSPX with Flu HA, HA and NA sequences)SEQ ID NO 8 GNLFIAPWGVIHHPHYEECSCYSEFAYGSFVRTVSLPVGADE (composite of Flu sequences of HA HA and NA with HSPXSEQ ID NO 9 HYEECSCYSEFAYGSFVRTVSLPVGADE (composite of Flu NA with HSPX)SEQ ID NO 10 SEFAYGSFVRTVSLPVGADEHYEECSCY(composite of Flu NA with HSPX)

Mice were immunized with ethanol killed MTB and MTB conjugate vaccineCRM-TB Pep01 according to standard protocol. The mice developed briskantibody titers to TB Pep01, mycolic acid, and other surface antigens asmeasured by ELISA (see Figures). Monoclonal antibodies were producedaccording to protocol, characterized and purified. Isolated MABs frommice immunized with ethanol killed MTB were generally type IgG1 whilethe conjugate CRM-Pep01 vaccine MABs were each IgG2 (Table 4). Thevaccines induced good serum titers to their respective immunogens. Bothmycolic acid binding MABs and MTB surface binding MABs were induced bywhole killed MTB. MABs to one or more immunity enhancing antigens arebelieved to useful for preventing and/or treating MTB or otherinfections. TB Pep02 induced serum titers to influenza and influenzapeptide (SEQ ID NO 5) and MABs were produced to the influenza peptidesequence (Table 4).

TABLE 4 Isolated and Purified Monoclonal Antibodies Vaccine Mouse MABIsotype Binding CRM-TB Pep01 1435 LD71BB2LD7 I IgG2a TB Pep01 BB2 CRM-TBPep01 1435 CA611GABCA6 IgG2b TB Pep01 II GA8 EtOH Killed 1323JG7111D3JG7 IgG1 MTB Surface MTB Lot 3 III D3 EtOH killed 1420 A891A5AB9I IgG1 MTB Surface MTB Lot 4 A5 GG911F2GG9 II IgG1 Mycolic Acid - F2 MTBSurface GG911F4GG9 II IgG1 Mycolic Acid - F4 Free GG911G2GG9 II IgG1Mycolic Acid - G2 MTB Surface CRM-TB Pep02 1438 FE1111A5FE11 IgG1Influenza II A5 Peptide (Seq 5) CRM-TB Pep02 1438 FE1111B3FE11 IgG1Influenza II B3 Peptide (Seq 5)

Example 6

Phagocytic cells (HL60) were incubated with ethanol killed MTB accordingto standard protocol. MTB were rapidly taken into the cells, butremained unchanged. In addition the phagocytic cells did not react. Inmarked contrast, the addition of a MAB (purified AB9IA5) that binds tothe surface of MTB caused a rapid and profound response in thephagocyte. The MTB was engulfed in vacuoles and the organism morphologywas rapidly destroyed. A fluorescent-based microscopy assay wasdeveloped to examine functional antibody activity against inactivatedMycobacterium tuberculosis (MTB) using differentiated HL60 cells in thepresence and/or absence of human complement. Bacteria: InactivatedMycobacterium tuberculosis was obtained from Battelle (West Jefferson,Ohio). Stock MTB: 1 mL bacterial suspensions fixed in EtOH orglutaraldehyde at a concentration between 1 and 10×10⁸ CFU/ml (OD600nM=1.000). Fixative removal: Ethanol and glutaraldehyde fixatives in MTBwere removed prior to staining and/or mixing with differentiated HL60cells to prevent damage to macrophages. Centrifugation: Fixativeremoval, staining, destaining and washing steps were done usingcentrifugation at 12000 rpm for 5 min, unless noted otherwise. Thelocation of the bacterial pellet was noted post centrifugation. Using apipette, ˜1,000 ul of supernatant from the tube was removed withoutdisrupting the pellet. The MTB pellet was resuspended with a maximumvolume of 1.2 mL per reagent and gently mixed by pipetting up and down4-5 times.

Procedure for Auramine O Staining of MTB

One ml of stock MTB was pelleted by centrifugation, washed 3 times withsterile tissue culture grade water to remove fixative. The MTB pelletwas resuspended with 1 mL of TB Auramine O and stained for 15 minutes atroom temperature and then washed once with demineralized water usingcentrifugation. The MTB pellet was resuspended with 1 mL TB Decolorizer(Truant-Moore) for 2-3 minutes then washed once with demineralizedwater, again using centrifugation. The MTB pellet was resuspended with 1mL TB Potassium Permanganate for 2-4 minutes and washed three times withdemineralized water using centrifugation. The MTB pellet was resuspendedin 1 mL sterile TC water. Growth and Differentiation of HL60 cells:Cells from the HL60 cell line (promyelocytic human leukemia cells:Ass#98070106, Lot 11D009; Sigma) were conditioned before use. StockHL60s: A frozen stock vial was thawed and expanded into a T-25 flask toa density of 5×10⁵ cells/mL in RPMI-1640 media containing 1% L-Glutaminesupplemented with 10% Fetal Bovine Serum (FBS). No antibiotics wereadded into the culture media. Undifferentiated HL60s: Cells were grownin 200 mL suspensions at 37° C. in a 5% CO₂ humidified atmosphere. Thecells were passaged every 3-4 days at 1-1.5×10⁵ cells/mL in RPMI-1640media containing 1% L-Glutamine and 10% FBS with no antibiotics.Differentiated HL60s: Cells were differentiated once a week at 2×10⁵cells/mL in RPMI-1640 media containing 1% L-Glutamine, 20% FBS, 1.25%Dimethyl Sulfoxide (DMSO) with no antibiotics. These cells were readyfor use in the assay at day 5 or 6 post induction with DMSO.Aseptically, one mL of differentiated HL60 cells at day 5 or 6 wasaliquoted into a microcentrifuge tube for use in the fluorescent-basedmicroscopy assay. ActinRed 555 Staining of Differentiated H160 cells:Differentiated HL60 cells were stained with ActinRed 555 Ready Probesreagent (Cat# R37112, Life Technologies). Two drops of ActinRed 555 dyewere added per mL of media/cells which were then gently vortexed andincubated for 5-15 minutes. Antibody Test Samples: Neat serum orpurified MAB samples were stored at minus 20° C. before use, thawed anddiluted in Phosphate Buffered Saline, pH 7.4 (Cat#100049, LifeTechnologies). The test samples selected had antibodies against MTB withtiters and/or binding activity confirmed by enzyme-linked immunosorbentassay (ELISA). Serum Dilution: Neat serum was diluted to a 1:100 testsample by adding 990 μL of PBS into a microcentrifuge tube followed with10 uL of neat serum into the 990 μL PBS. All was vortexed gently to mix.Purified MAB Dilution: One mL of MAB sample was prepared by diluting thestock MAB to 100 μg/mL in PBS. The 100 μg/mL sample was further dilutedto a 10 μg/mL by adding 45 μL of PBS into a microcentrifuge tube andadding 10 μL of purified MAB into the 90 μL PBS, again vortexing gentlyto mix. Complement: Human Complement was obtained from ThermofisherScientific, Cat NC988107; Lot 908634 and was aliquoted and stored atminus 80° C. until use. Stored sample was diluted in cold DMEM F-12media (Cat# D8062, Sigma) supplemented with Hepes Buffer (Cat# H0887,Sigma). Complement was diluted into a 1:16 sample by thawing in an icebath followed by the addition of 150 μL of cold media into amicrocentrifuge tube with 10 μL of human complement placed into the 150μL of cold media which was repeatedly pipetted to mix and kept in theice bath until use. With a Nikon Eclipse E600 Fluorescent Microscope,various combinations of test samples were examined that included: (1)ActinRed stained differentiated cells, (2) Auramine 0 stained MTB, (3)anti-MTB antibodies and (4) Human Complement. Individual or combinationsof samples were placed in labeled tubes as with the rations (see Table5): 100 μL HL60s: 100 μL MAB/Serum: 10 μL MTB: 10 μL C′. With a pipette,20 μL of sample were deposited into the middle of a micro slide andexamined using 100× magnification with emersion oil. The Nikon EclipseE600 Fluorescent Microscope Camera used a professional image acquisitionsoftware to process and manages images.

TABLE 5 FluMic 001 & 002 Slide/ Tube Number Test Sample Time point TS01HL60s only + ActinRed 555 0 min TS02 Inactivated MTB + Auramine O Stain0 min TS03 Differentiated HL60s + Inactivated MTB 3-60 min TS04Differentiated HL60s + Inactivated MTB 3-60 min anti-MTB/MAB A891A5 TS01Differentiated HL60s only + ActinRed 0 min 555 TS02 Inactivated MTB +Auramine O Stain 0 min TS03 Differentiated HL60s + Inactivated MTB 3-60min TS04 Differentiated HL60s + Inactivated 3-60 min MTB + anti-MTB MABGG911F2

Example 7 Antibody Stimulated Enhanced Phagocytic Activity

Studies were performed using HL 60 phagocytic cells to evaluate theability of antibodies to specific MTB target molecules to enhancephagocytic activity against MTB. Parallel studies using Group BStreptococci (GBS) demonstrated that antibodies directed against GBScapsule could facilitate rapid phagocytosis and killing of GBS by HL 60cells. Ethanol killed MTB was incubated in the absence of antibody withthe same conditioned HL 60 phagocytic cells. While the MTB was takeninside the phagocyte, the Bacillus remained normal in size andmorphology and the HL 60 cells were not stimulated and did not changeappearance. The MTB bacilli and HL 60 cells were both unchanged despitehaving the MTB in the cell cytoplasm. This has been considered to be aproblem for TB latency that MTB can persist unharmed inside phagocyticcells.

To analyze the ability of antibodies to specific MTB substances tostimulate phagocytes and enhance phagocytic activity, cloned andpurified mouse monoclonal antibodies (MAB) were used to various MTBtargets and epitopes (Table 4). Incubating MAB AB9 IA5 (Table 4) withMTB alone did appear to alter the shape or morphology of the bacillus.The halo zone around the bacillus (cell wall/surface matrix) wasunchanged. When HL 60 phagocytic cells were added to MTB and the MAB thecells were rapidly stimulated to engulf and phagocytize the bacilli,which appeared in vacuoles not in the cytoplasm. Over 3-10 minutes thevacuoles enlarged and bacillus morphology deteriorated. These changescontinued to progress over time with large blebs and protrusionsappearing throughout the cell. The MTB antibody enhanced phagocytosisand the bacillus up take and destruction visualized are consistent withthe phagocytosis and killing data demonstrated with antibody and GBS.The MAB AB9IA5 is an IgG1 antibody that binds to an unidentified MTBsurface antigen as determined by ELISA.

To further determine the ability of antibodies to stimulate phagocytesto engulf and destroy MTB, a different purified MAB GG9 II G2 (Table 4)was utilized that binds to a mycolic acid surface epitope as measured byELISA binding to both MTB bacilli and the mycolic acid moiety.Surprisingly when this MAB was incubated with MTB alone, the morphologychanged and the bacillus enlarged, with the cell wall/surface matrixhalo increasing in size. When HL 60 phagocytic cells were incubated withthe MTB and the MAB the phagocytes were markedly stimulated and extendedpseudopods that bound and engulfed the MTB. The pseudopods were activelymoving to bring the bacilli into vacuoles and over 5-15 minutes the MTBwas deformed and degraded. This anti-mycolic acid antibody promotedactive phagocytic engagement of MTB and stimulated profound up-take ofMTB and vacuole formation. Over the next several minutes the bacilliwere degraded and destroyed. Mycolic acid is a major component of thesurface matrix of MTB and considered to enable the MTB to be able toavoid effective phagocytosis and killing. Not all mycolic acidantibodies bind to the MTB bacillus (Table 4) and therefore will notstimulate phagocytes to engulf and kill MTB. This method of producingMABs that detect binding to whole MTB and target molecules and thenanalyzing the ability of the MAB to stimulate phagocytic HL 60 cellsusing fluorescent-based microscopy is useful for detecting MABs forpreventing or treating TB. In addition this method is useful forvalidating vaccine targets designed to induce antibodies to MTB.

Example 8

Purified MAB M1438 FEU II B3 was induced in a mouse by immunization withnon-natural, synthetically produced, MTB and Influenza (Flu) combinedpeptide antigen (Seq ID 7) that was conjugated to the CRM protein. Thiscombined peptide sequence contains 5 Flu peptides and one MTB peptide.Peptide 3 and Peptide 6 are non-natural Flu peptide composite epitopesof HA that combine the sequences of different Flu serotypes (Seq ID 2and 4). Pep 9 is a combined peptide of 3 and 6. Flu Pep 10 is a NApeptide that when synthesized with Pep 3 and 6 is sequence Pep 11 (SeqID 5 and 6). TB Pep 02 is a combination of TB Pep 01 (Seq ID 1) and FluPep 2, 3 and 4 (Seq ID 7). The MAB binding to various epitopes andantigens was analyzed by ELISA according to protocol (FIG. 7). The MABbound well to TB Pep 02 at both 1 and 10 μg/ml and at 10 μg/ml to FluPep 11 and surprisingly to gluteraldehyde killed MTB (Glut-K TB).Binding to Glut-K TB, but not to ethanol killed TB (EtOH-K TB)demonstrates that each type of microbial inactivation changes the normalantigens of the organism differently producing a variety of non-naturalantigens or epitopes and in this case ethanol and gluteraldehyde eachalter the surface moieties of MTB differently thereby creating new andnon-natural structures that are recognized by the immune system.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications and U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by referenceincluding U.S. Patent Application Publication No. 2013/0195909 entitled“Composite Antigenic Sequences and Vaccines” filed Jan. 25, 2013, U.S.Patent Application Publication No. 2011/0281754 entitled “Compositionsand Method for Detecting, Identifying and QuantitatingMycobacterial-Specific Nucleic Acid” filed Apr. 26, 2011, U.S. PatentApplication Publication No. 2009/0081202 entitled “ImmunogenicCompositions and Methods” filed Aug. 27, 2008, and U.S. ProvisionalApplication No. 61/746,962 entitled “Multipurpose Compositions forCollecting, Transporting and Storing Biological Samples” filed Dec. 28,2012. The term comprising, where ever used, is intended to include theterms consisting and consisting essentially of. Furthermore, the termscomprising, including, containing and the like are not intended to belimiting. It is intended that the specification and examples beconsidered exemplary only with the true scope and spirit of theinvention indicated by the following claims.

1. A vaccine for the treatment or prevention of infection ofMycobacterium tuberculosis (MTB) in a mammal comprising an antigen and apharmaceutically acceptable carrier, wherein the antigen comprises anon-naturally occurring MTB immunity enhancing antigen (IEA), whereinthe IEA is an altered form of a naturally occurring MTB antigen.
 2. Thevaccine of claim 1, wherein the IEA is derived from heat-killed MTB,alcohol-killed MTB, glutaraldehyde-killed MTB, or sonicated MTB.
 3. Thevaccine of claim 1, wherein the MTB immunity enhancing antigen comprisesan altered portion of an MTB heat shock protein, an MTB surface antigen,an MTB internal antigen, an MTB composite peptide, an MTB/Influenzaconjugate peptide, or peptide sequence listed in Table
 3. 4. The vaccineof claim 1, wherein the MTB immunity enhancing antigen generates ahumoral or cellular immune response in the mammal.
 5. The vaccine ofclaim 4, wherein the cellular immune response comprises phagocyticactivity.
 6. The vaccine of claim 4, wherein the period of time duringwhich the mammal remains protected again MTB infection is at least oneyear.
 7. The vaccine of claim 4, wherein the humoral immune responsecomprising the generation of antibodies that promote MTB phagocytosis.8. The vaccine of claim 1, wherein the pharmaceutically acceptablecarrier comprises oil, fatty acids, lipids, polymers, carbohydrates,gelatin, solvents, saccharides, buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents or an immunological inert substance, a carrier designated asgenerally recognized as safe (GRAS), or a combination thereof.
 9. Thevaccine of claim 1, further comprising an adjuvant.
 10. The vaccine ofclaim 9, wherein the adjuvant comprises alum, amino acids, proteins,lipid/water emulsion.
 11. The vaccine of claim 1, which containsmultiple different IEAs of MTB.
 12. A method for treating or preventingMTB infection comprising administering the vaccine of claim 1 to amammal.
 13. The method of claim 12, wherein the vaccine is administeredorally, by aerosol, intranasally, intramuscular, intravenously orsubcutaneously.
 14. The method of claim 12, wherein the vaccine isadministered before or after a primary vaccine.
 15. The method of claim14, wherein the vaccine is administered from about 2 to about 30 days inadvance of the primary vaccine.
 16. The method of claim 14, wherein theprimary vaccine is BCG, a killed whole-cell or attenuated MTB vaccine.17. The method of claim 12, wherein the vaccine generates a humoral orcellular immune response in the mammal.
 18. The method of claim 17,wherein the humoral immune response comprises generation of antibodiesspecifically reactive against mycolic acid of the surface of MTB. 19.The method of claim 17, wherein the cellular immune response comprisesenhanced activity of phagocytic cells.
 20. The method of claim 17,wherein the cellular immune response comprises generation of memory Tcells.
 21. A non-naturally occurring antibody that is specificallyreactive against an MTB moiety.
 22. The antibody of claim 21, whereinthe MTB moiety has been altered by chemical treatment.
 23. The antibodyof claim 22, wherein the chemical treatment is ethanol or gluteraldehydetreatment.
 24. The antibody of claim 21, wherein the MTB moiety isderived from an MTB heat shock protein, a peptidoglycan of MTB, amycolic acid of MTB, a lipoarabinomannan of MTB, or a combinationthereof.
 25. The antibody of claim 23, which is an IgA, IgD, IgE, IgG orIgM, or isolated Fab or Fc portions.
 26. The antibody of claim 21, whichcomprises a monoclonal or a polyclonal antibody.
 27. The monoclonalantibody of claim 26, which is derived from a mouse or a mouse-humanchimera, or is fully or partly humanized from a non-human antibody. 28.A hybridoma that produces the monoclonal antibody of claim
 26. 29. Amethod for treating or preventing an MTB infection in a mammal byadministering non-naturally occurring antibodies that enhancesphagocytic activity against MTB.
 30. The method of claim 29, wherein thephagocytic activity is enhanced in comparison to natural antibodiesgenerated against the whole organism.
 31. The method of claim 29,wherein the antibodies comprise polyclonal antibodies, monoclonalantibodies or both polyclonal and monoclonal antibodies.
 32. The methodof claim 29, wherein the antibodies are specifically reactive against anepitope of peptidoglycan, mycolic acid, lipoarabinomannan, or acombination thereof.
 33. The method of claim 29, wherein the antibodiesserve as an adjunctive therapy in combination with administration of anantibiotic.
 34. A method for treating or preventing an MTB infection ina mammal comprising administering a cocktail comprising two or moremonoclonal antibodies.
 35. The method of claim 34, wherein at least onemonoclonal antibody of the cocktail is specific for one or more of aheat shock protein, a peptidoglycan, a mycolic acid, alipoarabinomannan, peptidoglycan, of a mycolic acid, of alipoarabinomannan, or a combination thereof.
 36. The method of claim 34,wherein the cocktail contains multiple different monoclonal antibodies,each different monoclonal antibody specifically reactive against adifferent IEA.
 37. An antigen which comprises a non-naturally occurringimmunity enhancing epitope of MTB, wherein the immunity enhancingepitope is an altered form of a naturally occurring MTB epitope.
 38. Theantigen of claim 37, wherein the naturally occurring epitope is anepitope of a heat shock protein, peptidoglycan, mycolic acid,lipoarabinomannan, or a combination thereof.
 39. A method of identifyingone or more antibodies that activate phagocytizing cells, comprising:providing a microbe; generating antibodies that are specificallyresponsive to the microbe: incubating the generated antibodies with thephagocytizing cells; determining an activity of the phagocytizing cellsafter incubation with the antibodies; and selecting the one or moreantibodies that increase the activity of the phagocytizing cells ascompared to a control.
 40. The method of claim 39, wherein the microbeis live or killed MTB.
 41. The method of claim 39, wherein the microbeis treated with a chemical or physical agent.
 42. The method of claim41, wherein the chemical agent is ethanol or gluteraldehyde.
 43. Themethod of claim 39, wherein the antibodies are generated from a mouse ora human.
 44. The method of claim 39, wherein the antibodies aremonoclonal antibodies.
 45. The method of claim 39, wherein thephagocytizing cells are selected from the group consisting ofmacrophages, neutrophils, monocytes, mast cells, white blood cells,dendritic cells, phagocytic cell lines, HL-60 cells, U-937 cells, PMAtreated cells, PMA treated U-937 cells, and combinations thereof. 46.The method of claim 39, wherein the activity is cell proliferation, thepresence or absence of a marker, vacuole formation, or phagocyticactivity.
 47. The method of claim 39, wherein the activity is determinedby visual inspection, by antigen uptake, or by a fluorescent basedmicroscopy assay of the phagocytizing cells.
 48. The method of claim 39,wherein the phagocytizing cells show activity only on incubation withthe one or more selected antibodies.
 49. The method of claim 39, whereinthe control is the phagocytic activity of cells that have not beentreated with the antibodies, the phagocytic activity of cells afterincubation with antibodies provided against untreated microbes, or thephagocytic activity of cells after treatment with an agent that does notgenerate phagocytic activity.
 50. The method of claim 39, wherein theone or more antibodies selected treat or prevent a mammalian infectioncaused by the microbe.